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改良差时贴壁法分离培养鉴定小鼠骨髓间充质干细胞和内皮前体细胞
冯文磊 张猛 印双红 徐芳洁 王艳杰 陈雪玲 吴向未*
解剖学报 ›› 2015, Vol. 46 ›› Issue (2) : 282-288.
改良差时贴壁法分离培养鉴定小鼠骨髓间充质干细胞和内皮前体细胞
Isolation, cultivation and identification of mesenchymal stem cells and endothelial progenitor cells from murine bone marrow with a modified differential adhesion method
目的 探索同时从小鼠骨髓分离培养间充质干细胞(MSCs)与内皮前体细胞(EPCs)及对其鉴定的方法。方法 小鼠骨髓细胞经改良差时贴壁法分离，以48h为时间点，48h内贴壁细胞传至3代后行成骨、成软骨、成脂分化诱导实验，流式细胞术（FCM）检测其表面标记；48h后收集未贴壁细胞，传至3代后行血管形成实验，传至5代后行CD31免疫荧光细胞染色实验，FCM检测其表面标记。 结果 第3代48h内贴壁细胞可诱导分化为骨、软骨和脂肪细胞，FCM 检测Sca-1、CD29、CD45、CD11b 阳性率分别为（98.30±0.75）%,（97.47±1.32）%，（1.87±0.15）%，（1.03±0.71）%；第3代48h后贴壁细胞在基质胶上可形成血管样结构,第5代48h后贴壁细胞特异性表面抗原CD31呈阳性表达，FCM检测CD34、CD133、血管内皮生长因子受体（VEGFR2） 阳性率分别为（88.90±1.18）%，（92.73±2.90）%，（87.63±1.79）%。 结论 采用改良差时贴壁法可同时分离培养扩增小鼠骨髓 MSCs和 EPCs，且简便高效稳定可重复。
Objective To establish a method for simultaneously isolating, culturing and identifing of murine mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) from bone marrow. Methods The cells were isolated by a modified differential adhesion method from murine bone marrow and cultured for 48 hours. The primary adherent cells at 48 hours were cultured in LG-DMEM and the non-adhered cells were collected and induced by EGM-2MV complete medium in human fibronectin-coated dishes. Osteogenic, chondrogenic,and adipogenic induced multi-directional differentiation potentials were performed on the primary adherent cells, their immune phenotypes were detected by flow cytometry (FCM).Tube formation experiment on the matrigelin vitro and the expression of specific surface marker CD31 determined by immunofluoresence cell staining were identified for the subsequent adherent cells, and their immune phenotypes were detected by FCM.
Results The passage 3 of the primary adherent cells were identified by induced differentiation into osteoblasts, adipocytes and chondrocytes after induction. The expression levels of Sca-1, CD29, CD45, and CD11b were (98.30±0.75)%, (97.47±1.32 )% , (1.87±0.15)% and (1.03±0.71)% respectively. The passage 3 of the subsequent adherent cells were cultured on Matrigel, which resulted in the formation of tube-like structures.The expression of the passage 5 of the subsequent adherent cells specific surface marker CD31 was positive. The expression levels of CD34, CD133 and vascular endothelial growth factor receptor(VEGFR)2 were (88.90±1.18 )% , (92.73±2.90)%, and (87.63±1.79 )% respectively. Conclusions The modified differential adhesion is an efficient, stable, and replicable method that can simultaneously isolate and amplify mouse bone marrow MSCs and EPCs.
间充质干细胞 / 内皮前体细胞 / 骨髓 / 改良差时贴壁分离法 / 流式细胞术 / 小鼠
Mesenchymal stem cells / Endothelial progenitor cells / Bone marrow / Modified differential adhesion method / Cell culture / Flow cytometry / Mouse
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血管内皮前体细胞调控干细胞巢的实验研究
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